A genus of entomopathogenic Beaveria bassiana has been widely used in pest management, however little studies havebeen given to its virulence-related genes. To identify the roles of virulence genes, AtMT-induced random mutants weregenerated and followed by localization study with TAIL-PCR. Two genes, Complex I intermediated-associated protein30 (CIP30) and Autophagy protein 22 (Atg22), were predicted as virulence-related genes in B.bassiana JEF-007. To validatethe a possible relationship between two genes and fungal virulence, hpRNAi was performed. A hpRNAi plasmid wasconstructed as a model system to knock down of egfp gene in egfp-expressing B. bassiana transformant. A Real-TimePCR showed the knock down of egfp gene expression via hpRNAi. The CIP30 and Atg22 genes in B. bassiana willbe validated by the established RNAi technique. This work can be a strong platform for the functional genetics in B.bassiana.

Entomopathogenic fungi have been used as important part of integrated pest management program to control aphid. In particular, Beauveria bassiana was distributed throughout the world including temperate and tropical area, various habitats from alpine soil, desert soil to running water and both insect and plant. Especially the fungus has also been isolated from the surface and the interior of plants and act as natural control agent. Viability of fungi on the plant surface may be influenced by temperature, humidity, sunlight and plant type as well as fungal isolate. Persistence of treated fungal control agent on phylloplane and control efficacy may differ from environmental conditions and isolates. In this study, we investigated the persistence of an B. bassiana which is developing as prototype wettable powder to control cotton aphid, and the residual efficacy of the prototype on cucumber under three different greenhouse conditions.

Agrobacterium tumefaciens-mediated transformation (AtMT) has been widely used for generation of fungal transformants and recently applied to Beauveria bassiana. In this study to comprehend how the AtMT promoter influences on the expression of selection marker (hygromycin B resistance gene; hph), two different Ti-Plasmids were constructed: pCeg (gpdA promoter-based) and pCambia-egfp (CaMV 35S promoter-based). Putative transformants were subjected to the PCR, RT-PCR and qRT-PCR to inspect the T-DNA insertion rate and gene expression level. In conclusion, more than 80% of the colonies succeeded in AtMT transformation and the hph expression level of AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. This result can provide useful information on the AtMT of B. bassiana, especially antibiotics susceptibility and promoter-dependant expression level.

Negative effects of insecticides on non-target organism can be reduced by using selective and less toxic insecticides. We assessed the effect of subsequent application of flonicamid and Beauveria bassiana GHA on pests and natural enemies in soybean fields by recording their density in 6-8 d interval. Flonicamid controlled Aphis glycines Matsumura in 6 day after spray (DAS). Nymphs of Riptortus pedestris (F.) were also reduced by 46 and 48% in 12 and 19 DAS, respectively. Application of B. bassiana reduced R. pedestris nymph 50% in 16 DAS whereas adult R. pedestris, Medythia nigrobilineata Motschulsky and A. glycines were not significantly reduced in treated fields. Ladybird beetles, syrphid flies, hymenopteran wasps, and parasitism rate of R. pedestris eggs were not different between the treatment and control. These results suggest that flonicamid and B. bassiana showed less negative effect on the natural enemies and effective against aphids and nymphs stage of R. pedestris.

An entomopathogenic fungus, Beauveria bassiana ARP14, was isolated from field collected Riptortus pedestris (F.). Lethal concentration of ARP14 was compared with commercialized GHA strain against R. pedestris and its egg parasitoids Ooencyrtus nezarae Ishii and Gryon japonicum (Ashmead). Mortality and infection rate were evaluated in five concentrations, i.e., 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , and 1×10 5 conidia/mL using glass scintillation vial. The LC50 of 2nd, 4th instar, and adult stage of R. pedestris was not different between the two strains. The infection rate of ARP14 was higher than that of GHA in 4th instar and adult stage in 1×10 8 conidia/mL. The infection rate was low in ARP14 than GHA in both egg parasitoids. Therefore, our results suggest that the ARP14 strain can be used against R. pedestris with less negative effect on egg parasitoids than GHA strain.

Entomopathogenic fungi are potential pesticides as biological control agents. However, many previous studies have focused on basic researches to control pest using insect-pathogenic fungi. Herein, the molecular characteristics of insect defense mechanisms against fungal pathogens and fungal pathogenicity to insects were investigated. The whole genome of Beauveria bassiana (Bb) JEF-007 was sequenced (36,538,394 bp) using Pacbio sequencing. Based on this data, the transcriptomes of bean bug and fungus were analyzed using RNA-seq. In the transcriptome analyses, six and five genes were validated to be involved in the fungal pathogenicity and insect defense, respectively. This work can provide an understanding of the insect-pathogen interactions

Cotton aphid, Aphis gossypii Gloever is one of the major pests on a wide range of economically important crops in the world. The sustained use of chemical insecticides to control the aphid has led to the emergence of resistant strains to numerous used insecticides. As an alternative strategy entomopathogenic fungi have been used as part of integrated pest management program to control aphid, especially insecticide-resistance population. In particular, Beauveria bassiana-based commercial bio-insecticide has been used to reduce the pest population under greenhouse conditions in various countries. In this study, we investigated the control efficacy of a prototype of commercial mycopesticide using an B. bassiana (wettable powder) against cotton aphid on potted cucumber plant in greenhouse conditions.

Beauveria bassiana JEF-007 with strong virulence against Riptortus pedestiris was selected for the Agrobacerium tumefaciens-mediated transformation(AtMT). AtMT generated two transformants, B1-06 and C1-49, showed significantly reduced virulence against R. pedestris. To identify the virulence-related genes, thermal asymmetric interlaced(TAIL) PCR and flanking region analysis were performed. From the analysis, two genes, Complex I intermediated-associated protein 30(CIA30) and Autophagy protein 22(Atg22), possibly related virulence in B. bassiana JEF-007. For the analysis of two putative virulence-related genes in JEF-007, hairpin RNA interference (hpRNAi) is under consideration. This work can provide the functional roles of the virulence-related genes in B. bassiana JEF-007.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. To better understand the roles of Toll pathway in mealworm immune system, TmGPR and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGPR resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGPR. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2 to 6 days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, class A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS-A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while expression of class B CHS (CHS-B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS-A gene from the beet armyworm, Spodoptera exigua. The SeCHS-A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by RT-PCR analysis. Expression of SeCHS-A gene was suppressed by feeding double-stranded RNA (dsRNASeCHS-A, 150 ng/larva) in the fifth instar of S. exigua. The suppression of SeCHS-A gene expression significantly induced mortality on pupal stage. Also, larvae fed with dsRNASeCHS-A significantly enhanced pathogenicity of an entomopathogenic fungus, Beauveria bassiana ANU1. These results suggest that the SeCHS-A gene plays an important role in development of S. exigua and dsRNA, which is a specific to SeCHS-A gene, may be applied to effective pest control with B. bassiana.

Entomopathogenic fungi are natural enemies of insect pests and contribute to the natural regulation of their host populations. These fungal group are often used as active ingredients for microbial insect pest control. In addition, the potential antimicrobial effect by entomopathogenic fungi including Beauveria bassiana, Lecanicillium spp., and Isaria fumosorosea have recently been reported against fungal plant pathogens. Dual microbial control effects with entomopathogenic fungi against both aphids and cucumber powdery mildew had reported in Canada. In our previous studies we conducted bioassay with entomopathogenic fungi to develop dual microbial control agent which can control both aphid and fungal plant disease. We selected an Beauveria bassiana isolate which has high dual control effects against both cotton aphid, Aphis gossypii and sclerotinia rot, Sclerotinia sclerotiorum. In this study, we have tested the dual control efficacy of the B. bassiana isolate against cotton aphid and sclerotinia rot on whole potted cucumber plants. We found that the B. bassiana isolate protected the plant from cotton aphid and sclerotinia rot under laboratory condition.

A novel insect-infecting positive sense single-stranded RNA virus, Riptortus pedestris virus-1 (RiPV-1), was found in the Riptortus pedestris transcriptome data by de novo assembly and further confirmed by RACE method. The genome of RiPV-1 consists of 10,554 nucleotides (nt) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 10,371 nt encoding a 3,456 aa polyprotein and flanked by 71 and 112 nt 5' and 3' noncoding regions, respectively. RiPV-1 genome contains the consensus genome organization of picorna-like RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) array in that order from the 5' to the 3' end. From the phylogenetic analysis, RiPV-1 was clustered with unassigned insect RNA viruses, APV and KFV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses. Tissue tropism analysis revealed that RiPV-1 was relatively abundant in the thorax, abdomen, midgut and fat body. Interestingly, RiPV-1 replication was enhanced by Beauveria bassiana JEF-007 infection that was quantified using qRT-PCR. This study identified a novel insect-infecting virus and provided further insight into the relationship between virus, fungus and host.

Beauveria bassiana (Bb) is an entomopathogenic fungus with a wide host range, and is commonly used as an environment-friendly biopesticide. However, the molecular mechanisms of Bb-host interactions are not well understood. Here, RNA isolated from a highly virulent strain of B. bassiana (Bb JEF-007) and Riptortus pedestris (Hemiptera: Alydidae) (bean bug) infected with this strain were subjected to high throughput next generation sequencing (NGS) to analyze and compare transcriptomes. Differentially expressed gene (DEG) analysis showed that 2,381 genes were up-regulated and 2,303 genes were down-regulated upon infection. Most DEGs were classified into the categories of single-organism, cellular and metabolism processes by gene ontology (GO) analysis. Carbon metabolism-related enzymes in the glyoxylate cycle were significantly up-regulated, suggesting a possible role for them in Bb growth in the host. This work provides insight into how entomopathogenic B. bassiana occupies agriculturally harmful bean bug at the late stage, which might be essential during fungal infection.

Bean bug, Riptortus pedestris (Hemiptera: Alydidae) is an agriculturally serious pest in East Asian countries. Chemical pesticides have been contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Herein entomopathogenic Beauveria bassiana JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by contact-exposure and spray methods. The Agrobacterium tumefaciens-mediated transformation of B. bassiana JEF-007 generated random transformants and some mutants showed reduced virulence against Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and R. pedestris nymph. Compared to the wild-type, the two transformants showed remarkably different morphology, conidial production, and thermotolerance. To figure out pathogenicity-related genes, thermal asymmetric interlaced (TAIL) PCR of the random transformants was performed and possibly some virulence-related genes were predicted. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll signaling pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. TmGPR, antimicrobial peptide Tenecin 1 and Tenecin 2 were up-regulated after fungal infection. To better understand the roles of Toll signaling pathway in mealworm immune system, TmGRP and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 2 days post-dsRNA injection, and dramatically reduced at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGRP resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGRP. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

A mycoinsecticide, Beauveria bassiana (strain GHA) marketed as BotaniGard® ES, was evaluated as a biological control agent against Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) a major soybean pest. It was tested on commercial soybean fields with one time spray of both high (1×109) and medium (1×108spore/ml) concentration and double spray of the medium concentration. Nymph population decreased in all treated fields regardless of dose and frequency of spray. But, adult and egg populations were not affected. Alive nymphs collected from the fields and kept in laboratory showed infection rate ranging 35-68% in treated plots while 0% in control plots. It also reduced damage on soybean pods and seeds by 17.7-21.2 and 18.0-22.5% compare to control, respectively. Additionally, B. bassiana reduced other pests found in soybean fields including brown marmorated stink bug Halyomorpha halys (Stål) and two striped leaf beetle Medythia nigrobilineata Motschulsky. Also there was no negative effect on the parasitism of released non-viable eggs of R. pedestris. These results suggest that, in field condition, one time spraying of B. bassiana in the concentration of 1×108spore/ml can help reducing crop damage as well as R. pedestris nymph population.

Effectiveness of Beauveria bassiana ERL as entomopathogenic fungi against thrips pupae were evaluated on greenhouse cucumber(bumnong backdadagi), rose(Affinity) respectively. Control efficacy was calculated by the formula, (Dcontrol - Dtreatment)/Dcontrol x 100, where Dcontrol is the average density of thrips on the plots in which any pesticides and entomopathogenic fungi was not relesed and Dtreatment is the average density of thrips on the plots in which entomopathogenic fungi were released respectively. As a result, control efficacies of Beauveria bassiana ERL 10^7 cfu/g treatment against thrips on cucumber was 89.4~91.3%, respectively. In addition, control efficacy of Beauveria bassiana ERL 10^8 treatment against thrips on cucumber was 92.0~93.2%. Also, control efficacies of Beauveria bassiana ERL treatment against thrips on cucumber was 61.5-69.2%. Thus, even thrips control flow that occurs in the solanaceae vegetables thrips current period is officially Beauveria bassiana ERL 10 ^ 7 to effective treatment cfu / g, pepper and tomato cucumber and rose to occur will be effective. These results demonstrate the promise of entomopathogenic fungi, especially Beauveria bassiana ERL for management of thrips pupae. In addition to thrips control that is occurring in the solanaceae vegetables such as peppers and tomatoes will be effective.

Western flower thrips, (Pergande) (Thysanoptera: Thripidae) have been recognized as serious pests worldwide. Commercially available chemical pesticides challenges against the resistance of the pest and safety to consumers. Herein we investigated the usability of soil-treatment of entomopathogenic Beauveria bassiana (Bb) ERL isolates in tomatoes. Bb-egfp was highly virulent to western flower thrips in laboratory conditions and colonized in soil, which provided a chance for western flower thrips larvae to contact the colonized fungal mass. In a glasshouse trial, Bb ERL836 and Bb ERL1578 granule (GR) were applied to soil surface and mixed well, and five females were infested to each pot of tomato 3 days after the application. The two treatments significantly reduced thrips population in tomatoes 20 and 40 days post applications, comparable to the control efficacy of clothianidin granule (GR) formulation. In the following field trial, the two ERL fungal treatments showed 69.2% and 61.5% of control efficacy, which were similar to that of clothianidin GR (76.9%). This work suggests that applied B. bassiana colonized in soil well and thrips larvae easily contacted the fungal mass, which possibly enabled the control strategy to be alternative way to the chemical control.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. Supernatant showed different level of enzyme activity including chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence. TAIL-PCR of the random transformants revealed virulence-related genes. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.